Fluorescent probes, such as dyes, fluorescent proteins and quantum dots, have become indispensable tools in biomedical imaging, cell sorting, immuno-histology, high-throughput screening, and numerous other biochemical measurements. Although these luminescent probes are immensely useful, their relatively broad emission spectra, typically 30-100 nm, limit the number of probes that can be simultaneously used without ambiguity and often make their spectra indistinguishable from the background emission of endogenous molecules in tissues. Conventional fluorescence microscopes are equipped to resolve 3 to 4 dyes, and state-of-the-art cytometry is limited to eleven channels. Multiplexing four different dyes can give 16 (=24) combinations. Simultaneous expression of three genes encoding blue, green, and red fluorescent proteins at different ratios in cells, as in Brainbow and RGB marking, can generate hundreds of colors. However, the transfection is stochastic, and the fidelity of color reading is prone to noise. To date, the number of fluorescence colors for imaging has been limited to less than a dozen.
It is fundamentally challenging to engineer fluorophores for much narrower emission linewidth because of the quantum-mechanical broadening of the electronic levels in molecules. The irregular shapes and thermodynamic fluctuations resulted in spectral broadening of emission from semiconductor quantum dots. The attenuation of plasmonic electron oscillations in metallic nanoparticles resulted in emission widths of >50-100 nm. By comparison to these electronic resonance, optical resonance offers effective approaches to generate narrow emission lines. A laser is a great example. By placing fluorophores and semiconductor materials inside an optical cavity, an extremely narrow spectral line can be produced. The output of a single-frequency laser can be a millionth of nanometer in wavelength, tunable over the entire gain width by changing the cavity resonance.